The present study deals with the molecular genetics and biochemistry of two cyanobacterial toxins, cylindrospermopsin and saxitoxin. The molecular phylogenies of three putative cylindrospermopsin biosynthesis genes, encoding an amidinotransferase (aoaA), a non-ribosomal peptide synthetase (aoaB) and a polyketide synthase (aoaC) has been examined and functional modelling was carried out to gain insights into the mechanism of precursor recruitment in this pathway. An in vitro assay was developed to characterise biochemical requirements for saxitoxin biosynthesis, using enzyme extracts from Cylindrospermopsis raciborskii T3. All biosynthesis enzymes were located in the cytosol. The effects of substrates and inhibitors on the in vitro biosynthesis of saxitoxin were examined and predictions made regarding enzymes that may be involved. These predictions were used in a degenerate PCR approach to identify the saxitoxin biosynthesis gene cluster in cyanobacteria.
Bacterial fatty acid biosynthesis (FAS II) has been repeatedly validated as a drug target due to its lack of similarity to the mammalian fatty acid synthase system (FAS I). Acyl carrier protein (ACP) is a necessary cofactor in fatty acid biosynthesis, since all fatty acyl intermediates are covalently attached to it. We have solved the crystal structure of the E. coli enoyl-ACP reductase, FabI, in complex with its natural substrate, E. coli trans-2-dodecenoyl-ACP. The specific amino acid residues of FabI which are responsible for ACP binding have been identified and the conclusions from the crystallography analysis have been confirmed by mutagenesis and steady state kinetics studies.Due to reemergence of tuberculosis there is a need to develop new drugs. Triclosan is a potential lead compound for the development of new bacterial FASII inhibitors, specifically targeting InhA, which is the enoyl-ACP reductase from M. tuberculosis. We have determined that triclosan binds to InhA tighter when it is ionized.Additionally, we performed structure-activity studies of InhA inhibition by triclosan analogs.
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Systems biology is a relatively new biological study field that focuses on the systematic study of complex interactions in biological systems, thus using a new perspective (integration instead of reduction) to study them. Particularly from year 2000 onwards, the term is used widely in the biosciences, and in a variety of contexts. Systems biology is the study of the interconnected aspect of molecular, cellular, tissue, whole animal and ecological processes, and comprises mathematical and mechanistic studies of dynamical, mesoscopic, open, spatiotemporally defined, nonlinear, complex systems that are far from thermodynamic equilibrium.
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